CD317 maintains proteostasis and cell survival in response to proteasome inhibitors by targeting calnexin for RACK1-mediated autophagic degradation

Maintaining protein homeostasis (proteostasis) is crucial for cellular health. Cancer cells, due to intrinsic and extrinsic stresses, often exhibit imbalanced proteostasis networks, making them vulnerable to therapies targeting proteostasis regulators. Proteasome inhibitors (PIs), which disrupt the proteasome's protein degradation function, are a key example. While effective in some hematological malignancies, PI resistance frequently develops, underscoring the need to understand the mechanisms that preserve proteostasis in tumor cells. This study focuses on CD317 (also known as BST2, tetherin, or HM 1.24), a type II transmembrane glycoprotein overexpressed in various cancers. CD317 has been implicated in several cellular processes, including viral particle tethering, inflammation, immune modulation, and tumorigenesis, often promoting cell proliferation and drug resistance. Recent work suggested a potential role in ER stress regulation, hinting at a possible involvement in proteostasis. However, its precise contribution to proteostasis maintenance in tumor cells remained unclear. This research aims to investigate CD317's role in tumor cell proteostasis, particularly in the context of PI treatment. The study hypothesizes that CD317 plays a protective role against PI-induced proteostatic stress, and seeks to elucidate the underlying molecular mechanisms responsible for this effect. Understanding this mechanism could pave the way for developing strategies to overcome PI resistance.

The literature extensively documents the importance of proteostasis in cancer. Tumorigenesis is often associated with disrupted proteostasis due to factors like hypoxia, glucose deprivation, and oncogene activation. This disruption sensitizes cancer cells to drugs targeting proteostasis regulators, leading to the concept of "proteostasis addiction." Proteasome inhibitors (PIs) are established examples, targeting the proteasome – a key regulator of proteostasis responsible for degrading misfolded or damaged proteins. While PIs have shown efficacy in some cancers, especially hematological malignancies, the development of drug resistance remains a significant challenge. CD317's role in cancer is multifaceted, with its overexpression reported in several malignancies. Studies have demonstrated its involvement in promoting cell proliferation, migration, and resistance to various cytotoxic agents, including chemotherapy and immune-mediated cell killing. While a connection to ER stress has been hinted at, its precise role in proteostasis regulation hasn't been thoroughly investigated. This gap in knowledge provided the rationale for this study.

The study employed a multi-faceted approach combining bioinformatics analysis, cellular assays, and molecular biology techniques. **Bioinformatics:** CD317 expression levels were analyzed across multiple datasets (Oncomine and Cancer Cell Line Encyclopedia [CCLE]) for hematological malignancies. Survival analysis was performed using the Oncolnc database to assess the correlation between CD317 expression and patient prognosis. **Cell Culture and Transfection:** Several hematological malignancy cell lines (K562, Jurkat, NCI-H929) and HeLa cells were used. CD317 expression was manipulated using siRNAs and siRNA-resistant constructs. Other transfections included plasmids encoding tagged versions of calnexin and RACK1. **Intracellular Calcium Assay:** Intracellular calcium levels were measured using fluorescence staining with Fluo-4 AM and Fura-2 AM. ER calcium stores were assessed by measuring calcium release upon treatment with thapsigargin, an inhibitor of SERCA2b, the primary ER calcium pump. **Cell Death Assays:** Cell death was quantified using flow cytometry following staining with propidium iodide (PI) or Annexin V/PI. **qRT-PCR and Immunoblotting:** Gene expression levels were determined by qRT-PCR. Protein expression was analyzed using immunoblotting, and protein interactions were examined using co-immunoprecipitation (Co-IP). **RNA Sequencing:** RNA sequencing was performed on K562 cells to analyze changes in gene expression upon CD317 knockdown, with gene set enrichment analysis (GSEA) used to identify affected pathways. **Cycloheximide Chase Assay:** This technique was used to assess protein degradation rates. **LC-MS/MS:** LC-MS/MS was employed to identify CD317 binding partners. **Immunofluorescence and Microscopy:** Immunofluorescence staining was used to visualize the localization of proteins and assess colocalization. **Human Samples and Immunohistochemistry:** Immunohistochemistry (IHC) was performed on tissue microarrays to analyze CD317 and CNX expression in human T-cell lymphoma samples.

The study uncovered several key findings: 1. **CD317 Upregulation in Hematological Malignancies:** CD317 mRNA and protein levels were significantly elevated in various hematological malignancies compared to normal tissues, and high CD317 expression correlated with poor prognosis in acute myeloid leukemia (AML) patients. 2. **CD317's Protective Role against PI-Induced Cell Death:** Knockdown of CD317 in multiple hematological malignancy cell lines and solid tumor cell lines sensitized them to proteasome inhibitor-induced cell death. This effect was reversed upon re-expression of a siRNA-resistant CD317. 3. **CD317 Maintains Proteostasis:** CD317 knockdown resulted in increased accumulation of ubiquitinated proteins and ER stress markers, indicating impaired proteostasis. This effect was reversed by expressing siRNA-resistant CD317. 4. **CD317 Regulates Calcium Homeostasis:** CD317 knockdown led to a decrease in ER calcium levels and an increase in cytosolic calcium, disrupting calcium homeostasis. Re-expression of siRNA-resistant CD317 rescued this imbalance. 5. **CD317 Interacts with Calnexin (CNX):** LC-MS/MS and co-immunoprecipitation experiments identified CNX, an ER chaperone protein that regulates ER calcium levels, as a CD317 interacting partner. The interaction was found to be dependent on CD317's GPI anchor and was enhanced by autophagy inhibition. 6. **CD317 Targets CNX for Autophagic Degradation:** CD317 knockdown increased CNX protein levels by preventing its autophagic degradation. This was confirmed by experiments using autophagy inhibitors and activators and through colocalization studies of CNX and autophagosomes. 7. **RACK1 Mediates CD317's Effect on CNX:** RACK1, a component of the autophagy machinery, was identified as another CD317 interacting partner. CD317 facilitated the interaction between RACK1 and CNX, promoting CNX degradation. RACK1 knockdown abolished CD317's effect on CNX degradation. 8. **CNX is Essential for CD317's Function:** CNX knockdown in CD317-knockdown cells rescued the calcium imbalance, proteostasis defects, and increased susceptibility to PIs, demonstrating CNX's critical role in the CD317-mediated pathway.

The study's findings provide novel insights into the role of CD317 in maintaining proteostasis and cell survival in tumor cells under proteasome stress. The data clearly show that CD317 is a critical regulator of ER calcium homeostasis by targeting calnexin (CNX) for RACK1-mediated autophagic degradation. This mechanism, involving the interplay between CD317, CNX, RACK1, and autophagy, is vital for maintaining adequate ER calcium levels, which in turn favors protein folding and quality control. Disruption of this pathway, as seen upon CD317 knockdown, leads to ER stress, proteostasis collapse, and increased sensitivity to proteasome inhibitors. These findings have significant implications for cancer therapy. The upregulation of CD317 in hematological malignancies and its protective role against PI-induced cell death suggest that targeting CD317 could be a promising strategy to overcome PI resistance. Furthermore, the study elucidates a previously unrecognized function of autophagy in maintaining ER function and proteostasis, highlighting the complex interplay between different cellular pathways in ensuring proteome stability. Future research could explore the development of CD317 inhibitors as potential therapeutic agents or investigate the interaction between CD317 and other proteins involved in proteostasis.

This study identifies CD317 as a crucial regulator of proteostasis in tumor cells, highlighting a novel mechanism involving the interaction of CD317 with CNX and RACK1, ultimately regulating ER calcium homeostasis and promoting cell survival under proteasome inhibition stress. These findings suggest CD317 as a potential therapeutic target for overcoming PI resistance. Future research should focus on developing specific CD317 inhibitors and investigating the broader implications of this regulatory pathway in cancer.

The study primarily focused on hematological malignancy cell lines and a limited number of solid tumor cell lines. Further studies are needed to confirm these findings in a broader range of cancer types and in vivo models. The precise mechanisms underlying the CD317-CNX interaction and the involvement of other potential interacting partners could warrant further investigation. Finally, the clinical relevance of the identified pathway needs to be validated in larger clinical trials.